U.S. patent application number 15/781737 was filed with the patent office on 2018-12-20 for method for purifying fibrinogen.
This patent application is currently assigned to GREEN CROSS HOLDINGS CORPORATION. The applicant listed for this patent is GREEN CROSS HOLDINGS CORPORATION. Invention is credited to Hyo Jin KIM, Jun Sic KIM, Ju Ho LEE, Ji Yoon PARK, Yong Won SHIN, Jae Woon SON.
Application Number | 20180362615 15/781737 |
Document ID | / |
Family ID | 59311943 |
Filed Date | 2018-12-20 |
United States Patent
Application |
20180362615 |
Kind Code |
A1 |
KIM; Jun Sic ; et
al. |
December 20, 2018 |
METHOD FOR PURIFYING FIBRINOGEN
Abstract
A method for purifying fibrinogen includes steps of: (a)
precipitating fibrinogen of a fibrinogen-containing solution by
adding glycine to the solution for a concentration of glycine to be
1.5 to 2.5M, and then removing a supernatant and recovering a
precipitate (1.sup.st glycine precipitation); (b) dissolving the
precipitate of 1.sup.st glycine precipitation of step (a) in a
dissolution buffer to obtain a solution, precipitating the solution
by adding glycine thereto for a concentration of glycine to be 0.2
to 1.2M, and recovering a supernatant (2.sup.nd glycine
precipitation); (c) precipitating the supernatant of step (b) by
adding glycine thereto for a concentration of glycine to be 1.5 to
2.5M, and recovering a precipitate (3.sup.rd glycine
precipitation); and (d) dissolving the precipitate of step (c) in a
dissolution buffer to obtain a solution, and subjecting the
solution to nanofiltration using a nanofilter.
Inventors: |
KIM; Jun Sic; (Yongin-si,
KR) ; KIM; Hyo Jin; (Yongin-si, KR) ; PARK; Ji
Yoon; (Yongin-si, KR) ; LEE; Ju Ho;
(Yongin-si, KR) ; SON; Jae Woon; (Yongin-si,
KR) ; SHIN; Yong Won; (Yongin-si, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GREEN CROSS HOLDINGS CORPORATION |
Yongin-si, Gyeonggi-do |
|
KR |
|
|
Assignee: |
GREEN CROSS HOLDINGS
CORPORATION
Yongin-si, Gyeonggi-do
KR
|
Family ID: |
59311943 |
Appl. No.: |
15/781737 |
Filed: |
January 12, 2017 |
PCT Filed: |
January 12, 2017 |
PCT NO: |
PCT/KR2017/000389 |
371 Date: |
June 6, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 1/36 20130101; C07K
1/30 20130101; C07K 14/75 20130101; A61K 38/36 20130101; C07K 1/34
20130101; B01J 20/08 20130101 |
International
Class: |
C07K 14/75 20060101
C07K014/75; B01J 20/08 20060101 B01J020/08; C07K 1/30 20060101
C07K001/30; C07K 1/34 20060101 C07K001/34; C07K 1/36 20060101
C07K001/36 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 12, 2016 |
KR |
10-2016-0003475 |
Claims
1. A method for purifying fibrinogen, comprising the steps of: (a)
precipitating fibrinogen of a fibrinogen-containing solution by
adding glycine to the solution for a concentration of glycine to be
1.5 to 2.5M, and then removing a supernatant and recovering a
precipitate (1.sup.st glycine precipitation); (b) dissolving the
precipitate of 1.sup.st glycine precipitation of step (a) in a
dissolution buffer to obtain a solution, precipitating the solution
by adding glycine thereto for a concentration of glycine to be 0.2
to 1.2M, and recovering a supernatant (2.sup.nd glycine
precipitation); (c) precipitating the supernatant of step (b) by
adding glycine thereto for a concentration of glycine to be 1.5 to
2.5M, and recovering a precipitate (3.sup.rd glycine
precipitation); and (d) dissolving the precipitate of step (c) in a
dissolution buffer to obtain a solution, and subjecting the
solution to nanofiltration (NF) using a nanofilter.
2. The method of claim 1, wherein the dissolution buffer used for
dissolving the precipitate in steps (b) and (d) comprises 10 to 100
mM sodium citrate of pH 6.0 to 9.0.
3. The method of claim 1, wherein the nanofiltration in step (d) is
performed at 20 to 37.degree. C. under a pressure of 0.5 to 2.5
bar.
4. The method of claim 1, wherein the fibrinogen-containing
solution of step (a) is obtained by a process comprising the steps
of: (i) dissolving a fibrinogen-containing cryopaste using a
dissolution buffer to obtain a solution; (ii) adding an adsorbent
to the solution, thereby removing impurities by adsorption; and
(iii) inactivating viruses, contained in the cryopaste, by
solvent/detergent (S/D) treatment.
5. The method of claim 4, wherein the cryopaste in step (i) is
dissolved using the dissolution buffer at a volume ratio of 1:2 to
1:6.
6. The method of claim 4, wherein the dissolution buffer in step
(i) is a buffer comprising 20 mM sodium citrate, 100 mM NaCl, and
100 mM glycine.
7. The method of claim 4, wherein the adsorbent used in step (ii)
is aluminum hydroxide (Al(OH).sub.3).
8. The method of claim 7, wherein the aluminum hydroxide is used in
an amount of 0.05 to 0.5 wt % based on the weight of the
cryopaste.
9. The method of claim 4, wherein the solvent/detergent treatment
of step (iii) is performed using a solvent/detergent solution
comprising a non-ionic detergent and tri-n-butyl phosphate
(TNBP).
10. The method of claim 9, wherein the non-ionic detergent is at
least one selected from the group consisting of polysorbate 80
(Tween 80), polysorbate 20 (Tween 20), Triton X-100, and Triton
X-45.
11. The method of claim 1, further comprising, between steps (c)
and (d), a step (c') dissolving the precipitate recovered in step
(c) in dissolution buffer to obtain a solution, precipitating the
solution by adding glycine thereto for a glycine concentration to
be identical to the glycine concentration of step (a), to obtain a
precipitate (4.sup.th glycine precipitation).
12. The method of claim 1, further comprising, after the
nanofiltration of step (d), at least one step selected from among
(e) ultrafiltration/diafiltration (UF/DF) step; and (f) formulation
step.
Description
TECHNICAL FIELD
[0001] The present disclosure relates to a method for purifying
fibrinogen, and more particularly, to a method for purifying
fibrinogen, comprising the steps of: (a) precipitating fibrinogen
of a fibrinogen-containing solution by adding glycine to the
solution for a concentration of glycine to be 1.5 to 2.5M, and then
removing a supernatant and recovering a precipitate (1.sup.st
glycine precipitation); (b) dissolving the precipitate of 1.sup.st
glycine precipitation of step (a) in a dissolution buffer to obtain
a solution, precipitating the solution by adding glycine thereto
for a concentration of glycine to be 0.2 to 1.2M, and recovering a
supernatant (2.sup.nd glycine precipitation); (c) precipitating the
supernatant of step (b) by adding glycine thereto for a
concentration of glycine to be 1.5 to 2.5M, and recovering a
precipitate (3.sup.rd glycine precipitation); and (d) dissolving
the precipitate of step (c) in a dissolution buffer to obtain a
solution, and subjecting the solution to nanofiltration (NF) using
a nanofilter.
BACKGROUND ART
[0002] Fibrinogen, also known as clotting factor I, plays a key
role in haemostasis and wound healing. It is a glycoprotein
synthesized in the liver with an apparent molecular weight of 340
kDa, and is composed of two dimers, each of them built of three
pairs of non-identical polypeptide chains called A.alpha., B.beta.
and .gamma. linked by disulfide bridges. It circulates in the blood
stream at a concentration of approximately 150-400 .mu.g/ml. Upon
injury of blood vessels, blood platelets are activated and a plug
is formed. Fibrinogen is involved in primary haemostasis by aiding
cross-linking of activated platelets.
[0003] At the same time, activation of the clotting cascade is
initiated. As the endpoint, fibrinogen is converted into fibrin by
proteolytic release of fibrinopeptide A and, at a slower rate,
fibrinopeptide B by thrombin. The soluble fibrin monomers are
assembled to double stranded twisted fibrils. Subsequently, these
fibrils are arranged in a lateral manner, resulting in thicker
fibers. These fibers are then cross-linked by FXIIIa to a fibrin
network, which stabilizes the platelet plug by interactions of the
fibrin with activated platelets, resulting in a stable clot.
[0004] Disorders and Deficiencies
[0005] Congenital afibrinogenaemia is a rare bleeding disorder,
where patients are suffering from inadequate clotting of the blood
due to the lack or malfunction of fibrinogen. This disorder might
lead to spontaneous bleeding or excessive bleeding after minor
traumata or during interventional procedures.
[0006] Acquired deficiencies in fibrinogen are much more common
than congenital afibrinogenaemia and may be induced by
haemodilution or other events such as blood losses during surgery,
traumata, disseminated intravascular coagulation (DIC) or
sepsis.
[0007] Fibrinogen deficiencies can be corrected to normal
fibrinogen levels of about 1.5-3 g/l in plasma by replacement
therapy with intravenous infusion of fresh frozen plasma or
cryoprecipitate. However, these treatments are afflicted with the
risk of introduction of pathogens, e.g. viruses or prions, into a
patient, and thus cause additional disorders. It is thus advisable
to intravenously apply virus inactivated fibrinogen compositions to
restore fibrinogen at physiological levels in a safe way.
[0008] Fibrinogen Purification
[0009] Human plasma contains a complex mixture of more than 100
proteins, with fibrinogen accounting for about 2% of the total
amount of protein. Therefore, the purification and isolation of
fibrinogen usually requires a plurality of steps, and diverse
combinations are possible for these individual process steps.
[0010] One important component of the purification of fibrinogen
from human plasma is conventionally precipitation. Known
precipitation methods use amino acids such as glycine or alanine
(see: European Patent No. EP0383234; International Publication No.
WO2001/48016; Jakobsen & Kierulf, Thrombosis Research,
3:145-159, 1973), ammonium sulfate (see: U.S. Pat. No. 5,773,033;
U.S. Pat. No. 6,037,457; Takeda, Y, Journal of Clinical
Investigation, 45:103-111, 1966), polymers such as polyethylene
glycol (PEG) (see: International Publication No. WO1995/25748; Vila
et al., Thrombosis Research 39:651-656, 1985), ethanol (see:
European Patent No. EP0408029 wherein fibrinogen is precipitated
and separated from other plasma proteins with 5-10% ethanol;
Blomback & Blomback, Arkiv For Kemi, 10:415-443, 1956),
sulphated polysaccharides (SPS, e.g., heparin) (see: International
Publication No. WO1999/37680; U.S. Pat. No. 4,210,580), and
solutions of low ionic strength (see: U.S. Pat. No. 4,188,318).
[0011] Examples of the disclosed conventional technologies
associated with glycine precipitation in human fibrinogen
purification processes include a two-step glycine precipitation
process which comprises removing impurities produced in a
pasteurization process for removing pathogenic particles (e.g.,
virus) and concentrating fibrinogen (CSL Behring, 2009), and a
three-step glycine precipitation process obtaining a fibrinogen
precipitate by use of only high-concentration 1.5-1.7M glycine (For
a reference: U.S. Pat. No. 7,442,308; Grifols, E S). However, it
has not been reported yet that a glycine precipitate containing
impurities and insoluble substances is removed by performing a
precipitation process using glycine at a concentration which is 2
to 4 times lower than the concentration of glycine used in the
above-described process, the recovered supernatant is precipitated
with a high concentration of glycine, and the recovery rate of
high-purity fibrinogen is increased by improving the efficiency of
nanofiltration, when a fibrinogen solution containing the
precipitate dissolved therein is nanofiltered.
[0012] Under this technical background, the present inventors have
made extensive efforts to develop a method for purifying
fibrinogen, comprising the steps of: (a) precipitating fibrinogen
of a fibrinogen-containing solution by adding glycine to the
solution for a concentration of glycine to be 1.5 to 2.5M, and then
removing a supernatant and recovering a precipitate (1.sup.st
glycine precipitation); (b) dissolving the precipitate of 1.sup.st
glycine precipitation of step (a) in a dissolution buffer to obtain
a solution, precipitating the solution by adding glycine thereto
for a concentration of glycine to be 0.2 to 1.2M, and recovering a
supernatant (2.sup.nd glycine precipitation); (c) precipitating the
supernatant of step (b) by adding glycine thereto for a
concentration of glycine to be 1.5 to 2.5M, and recovering a
precipitate (3.sup.rd glycine precipitation); and (d) dissolving
the precipitate of step (c) in a dissolution buffer to obtain a
solution, and subjecting the solution to nanofiltration (NF) using
a nanofilter, the efficiency of filtration increases at least
2-fold, so that high-purity fibrinogen from which virus was removed
can be recovered, thereby completing the inventions of the present
disclosure.
DISCLOSURE OF INVENTION
Technical Problem
[0013] It is an object of the present disclosure to provide a
method for purifying high-purity fibrinogen, which can increase the
efficiency of nanofiltration.
Technical Solution
[0014] To achieve the above object, the present disclosure provides
a method for purifying fibrinogen, comprising the steps of: (a)
precipitating fibrinogen of a fibrinogen-containing solution by
adding glycine to the solution for a concentration of glycine to be
1.5 to 2.5M, and then removing a supernatant and recovering a
precipitate (1.sup.st glycine precipitation); (b) dissolving the
precipitate of 1.sup.st glycine precipitation of step (a) in a
dissolution buffer to obtain a solution, precipitating the solution
by adding glycine thereto for a concentration of glycine to be 0.2
to 1.2M, and recovering a supernatant (2.sup.nd glycine
precipitation); (c) precipitating the supernatant of step (b) by
adding glycine thereto for a concentration of glycine to be 1.5 to
2.5M, and recovering a precipitate (3.sup.rd glycine
precipitation); and (d) dissolving the precipitate of step (c) in a
dissolution buffer to obtain a solution, and subjecting the
solution to nanofiltration (NF) using a nanofilter.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows a method for purifying fibrinogen according to
the present disclosure.
[0016] FIG. 2 shows a specific preparation method for formulating a
fibrinogen product from cryopaste by use of a method for purifying
fibrinogen according to the present disclosure.
[0017] FIG. 3 shows the purity of fibrinogen obtained when
performing only 1.sup.st and 3.sup.rd glycine precipitation
processes without a 2.sup.nd glycine precipitation process.
[0018] FIG. 4 shows the purity of fibrinogen obtained when
performing all the 1.sup.st to 3.sup.rd glycine precipitation
processes (under the conditions of 20.degree. C., 0.75M and 15
mg/mL).
[0019] FIG. 5 shows the purity of fibrinogen obtained when
performing all the 1.sup.st to 3.sup.rd glycine precipitation
processes (under the conditions of 25.degree. C., 0.8M and 20
mg/mL).
[0020] FIG. 6 shows the purity of fibrinogen obtained when
performing all the 1.sup.st to 3.sup.rd glycine precipitation
processes (under the conditions of 15.degree. C., 0.8M and 10
mg/mL).
[0021] FIG. 7 shows the purity of fibrinogen obtained when
performing all the 1.sup.st to 3.sup.rd glycine precipitation
processes (under the conditions of 15.degree. C., 0.5M and 20
mg/mL).
[0022] FIG. 8 shows the amount of fibrinogen filtered through a
Planova 20N filter after a 2.sup.nd glycine precipitation process
was performed or not performed.
BEST MODE FOR CARRYING OUT THE INVENTION
[0023] Unless defined otherwise, all the technical and scientific
terms used herein have the same meaning as those generally
understood by one of ordinary skill in the art to which the
invention pertains. Generally, the nomenclature used herein and the
experiment methods, which will be described below, are those well
known and commonly employed in the art.
[0024] The terms "process", "purification", "separation" and
"isolation", as used interchangeably herein, refer to the use of at
least one method or system to achieve a specific purpose (for
example, fibrinogen purification) in a purification process.
[0025] In one aspect, the present disclosure is directed to a
method for purifying fibrinogen, comprising the steps of: (a)
precipitating fibrinogen of a fibrinogen-containing solution by
adding glycine to the solution for a concentration of glycine to be
1.5 to 2.5M, and then removing a supernatant and recovering a
precipitate (1.sup.st glycine precipitation); (b) dissolving the
precipitate of 1.sup.st glycine precipitation of step (a) in a
dissolution buffer to obtain a solution, precipitating the solution
by adding glycine thereto for a concentration of glycine to be 0.2
to 1.2M, and recovering a supernatant (2.sup.nd glycine
precipitation); (c) precipitating the supernatant of step (b) by
adding glycine thereto for a concentration of glycine to be 1.5 to
2.5M, and recovering a precipitate (3.sup.rd glycine
precipitation); and (d) dissolving the precipitate of step (c) in a
dissolution buffer to obtain a solution, and subjecting the
solution to nanofiltration (NF) using a nanofilter.
[0026] Unlike conventional process comprising a step of obtaining a
precipitate from a precipitation process using glycine, the method
for purifying fibrinogen according to the present disclosure, which
comprises a precipitation process performed using glycine at a
relatively low concentration, and then the supernatant, but not the
precipitate, is recovered and nanofiltered, makes it possible to
obtain fibrinogen with very high purity. Specifically, the
fibrinogen purification method according to the present disclosure
has effects in that the use of the inventive method increases the
filtration efficiency of a nano-filter by more than twice to
exhibit a high fibrinogen recovery with a purity of 98%, and the
precipitation processes are performed using glycine at lowered
concentrations so that impurities produced in the virus
inactivation process can be removed as well as the filtration
efficiency of the nano-filter can be increased as shown in Table 2
and FIGS. 3 to 8.
[0027] In an embodiment, the glycine concentration in the 1.sup.st
glycine precipitation in step (a) is 1.5 to 2.5M, preferably 1.8 to
2.2M, most preferably 1.9 to 2.1M.
[0028] In an embodiment, the glycine concentration in the 2.sup.nd
glycine precipitation in step (b) is 0.2 to 1.2M, preferably 0.3 to
1.1M, most preferably 0.5 to 0.9M.
[0029] In an embodiment, the glycine concentration in the 3.sup.rd
glycine precipitation in step (c) is 1.5 to 2.5M, preferably 1.8 to
2.3M, most preferably 1.9 to 2.2M.
[0030] The dissolution buffer used for dissolving the precipitate
in steps (b) and (d) may be a dissolution buffer used commonly in
the art to which the present disclosure pertains, and the
dissolution of the precipitate may be performed using a buffer
containing preferably 10 to 100 mM, more preferably 30 to 70 mM of
sodium citrate at pH 6.0 to 9.0, preferably 7.0 to 8.0, most
preferably 7.5.
[0031] In an embodiment, the glycine precipitation and the recovery
of the precipitate may be performed by adding a suitable
concentration of glycine in each step, and stirring with 200 to
1,000 rpm at 4 to 30.degree. C., followed by centrifugation under
3,000 to 10,000 rpm at 4 to 25.degree. C., but the scope of the
present disclosure is not limited thereto.
[0032] In addition, the nanofiltration in step (d) may be performed
using a commercially-available nanofiltration system, and the type
of the filter that can be used in the present disclosure is
preferably SV4 20N from Pall Corporation or Planova 20N from Asahi
Kasei Co., Ltd., but is not limited thereto, and may be performed
using a suitable buffer. Preferably, a buffer containing 10 to 30
mM sodium citrate, 50 to 150 mM NaCl, 1 to 7%, preferably 2 to 5%
arginine at pH 6.0 to 9.0, preferably 7.0 to 8.0, most preferably
7.2 to 7.8 might be used. The nanofiltration in step (d) may
performed at 20 to 37.degree. C., preferably 28 to 35.degree., most
preferably 25 to 35.degree. under a pressure of 1 to 2 bar when
using SV4 20N and 0.5 to 1 bar when using Planova 20N.
[0033] Preferably, the fibrinogen-containing solution of step (a)
may be obtained by a process comprising the steps as follows, but
is not limited thereto:
[0034] (i) dissolving a fibrinogen-containing cryopaste using a
dissolution buffer to obtain a solution;
[0035] (ii) adding an adsorbent to the solution, thereby removing
impurities by adsorption; and
[0036] (iii) inactivating viruses, contained in the cryopaste, by
solvent/detergent (S/D) treatment.
[0037] The cryopaste in step (i) may be dissolved using the
dissolution buffer at a volume ratio of 1:2 to 1:6, preferably 1:3
to 1:5, most preferably 1:4. The dissolution buffer in step (i) may
be a buffer containing 10 to 30 mM, preferably 20 mM sodium
citrate, 50 to 150 mM, preferably 100 mM NaCl, and 50 to 150 mM,
preferably 100 mM glycine, but is not limited thereto.
[0038] In addition, the adsorbent used in step (ii) is preferably
aluminum hydroxide (Al(OH).sub.3), but is not limited thereto. The
Al(OH).sub.3 adsorption gel which is an impurity-adsorbed
adsorption precipitate may be removed from centrifugation. The
adsorbent, aluminum hydroxide may have a concentration of 1.0 to
5.0%, preferably 1.5 to 3.0%, most preferably 2.0%, and may be used
in an amount of 0.05 to 0.5 wt %, preferably 0.07 to 0.3 wt %, most
preferably 0.9 to 0.15 wt % based on the weight of the
cryopaste.
[0039] In the solvent/detergent treatment of step (iii), any
solvent and detergent may be used without limitations, as long as
they have the capability to inactivate viruses, particularly lipid
enveloped viruses. The detergent may be selected from the group
consisting of non-ionic and ionic detergents and is preferably
selected to be substantially non-denaturing. Particularly, a
non-ionic detergent is preferable in terms of removal convenience,
and the solvent is most preferably tri-n-butyl phosphate (TNBP) as
disclosed in U.S. Pat. No. 4,764,369, but is not limited
thereto.
[0040] Preferably, the solvent/detergent treatment of step (iii)
may be performed using a solvent/detergent solution containing a
non-ionic detergent and tri-n-butyl phosphate (TNBP). The
virus-inactivating agent that is used in the present disclosure is
preferably a mixture of TNBP and at least one selected from among
polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), Triton X-100,
and Triton X-45, but is not limited thereto.
[0041] The preferred solvent/detergent mixture is added for the
concentration of TNBP in the fibrinogen-containing solution to be
0.2 to 0.6 wt %, preferably 0.24 to 0.36 wt %, and for the
concentration of Tween 80 to be 0.5 to 1.5 wt %, preferably 0.8 to
1.2 wt %.
[0042] In addition, the method of the present disclosure may
further comprise, between steps (c) and (d), step (c') of
dissolving the precipitate recovered in step (c) in dissolution
buffer to obtain a solution, precipitating the solution by adding
glycine thereto for a glycine concentration to be identical to the
glycine concentration of step (a), to obtain a precipitate
(4.sup.th glycine precipitation).
[0043] The method of the present disclosure may optionally
comprise, after the nanofiltration of step (d),
[0044] (e) ultrafiltration/diafiltration (UF/DF) or ultrafiltration
step; and/or
[0045] (f) formulation step.
[0046] As used herein, "diafiltration" refers a technique that
removes or collects any component (e.g., particles) from a target
substance (solution) using a permeable filter capable of achieving
separation according to the molecular weight (molecular size) of
the component, thereby increasing the purity of the target
substance. The ultrafiltration/diafiltration (UF/DF) in step (e)
may be performed using a typical UF/DF system, and may comprise a
change to a constant osmotic pressure, an exchange of a buffer, and
an adjustment of the concentration of the buffer.
[0047] The term "fibrinogen" preferably refers human fibrinogen
which can be purified, for example, from a mixture which contains
fibrinogen and has been obtained from human blood. The phrase
"mixture obtained from blood" refers, for example, whole blood,
blood plasma, plasma fractions or plasma precipitates. Fibrinogen
from human plasma or cryoprecipitate, particularly fibrinogen from
Cohn fraction 1, is preferred. Fibrinogen can be isolated both from
pooled plasma donations and from individual donations.
[0048] Human fibrinogen can also be obtained from the body fluids
(e.g., milk) of transgenic animals (U.S. Pat. No. 5,639,940) or by
recombinant expression from cell culture (U.S. Pat. No. 6,037,457).
In addition, fibrinogen can be isolated from the appropriate
fermentation supernatants or fractions produced therefrom.
Meanwhile, fibrinogen can be isolated from animal blood containing
fibrinogen, preferably blood derived from mammals (e.g., pigs,
horses, cows, goats, sheep, and dogs).
[0049] Impurities or insoluble proteins in the present disclosure
refer all proteins which occur in plasma other than fibrinogen or
appear in the body fluids (e.g., milk) of transgenic animals or in
the cell culture supernatant, and preferably, fibrinogen-degrading
proteins which are able to degrade fibrinogen by proteolysis, or
precursors of fibrinogen-degrading proteins (proenzymes) which must
be previously activated for proteolytic degradation of fibrinogen,
or activators of fibrinogen-degrading proteases, and fibrinogen
degradation fragments having low molecular weights might be
included.
[0050] The phrase "fibrinogen-containing cryopaste" refers a
fibrinogen-containing cryoprecipitate derived from the
above-described animals (preferably humans).
[0051] In the present disclosure, the "pH" of a solution (e.g.,
dissolved solution) measures the acidity or alkalinity relative to
the ionization of a water sample. The pH of water is neutral, i.e.,
7. Most pH readings range from 0 to 14. Solutions with a higher
[H+] than water (pH less than 7) are acidic; solutions with a lower
[H+] than water (pH greater than 7) are basic or alkaline. pH can
be measured using a pH meter. pH of buffer may be adjusted using an
acid or base such as HCl or NaOH. The dissolution buffer used in
the present disclosure is preferably in the neutral pH range.
[0052] As used herein, the term "purification" can be
interchangeably used with the term "clarification" and refers
re-dissolving a precipitate or the like using a buffer, and then
removing impurities from the solution resulting from the
re-dissolution.
[0053] The fibrinogen purified by the method of the present
disclosure has a purity of at least 90%, preferably at least 93%,
most preferably at least 98%. Particularly, the produced fibrinogen
protein in the present disclosure may be a fibrinogen protein
having a purity of at least 98%.
EXAMPLES
[0054] Hereinafter, the present disclosure will be described in
further detail with reference to examples. It will be obvious to a
person having ordinary skill in the art that these examples are for
illustrative purposes only and are not to be construed to limit the
scope of the present disclosure.
Example 1: Process for Dissolution of Fibrinogen-Containing
Cryopaste and Treatment with Al(OH).sub.3 Gel
[0055] 1) 4 L of dissolution buffer (containing 50 mM sodium
citrate at pH 7.5, 100 mM sodium chloride (NaCl), and 100 mM
glycine) for dissolving fibrinogen-containing cryopaste was placed
in a jacketed beaker, and then stirred at a controlled circulator
temperature of 37.degree. C. for 30 minutes or more. At this time,
the temperature of the buffer was maintained at 37.degree. C.
[0056] 2) 1 kg of fibrinogen-containing cryopaste was added to 4 L
of the dissolution buffer (fibrinogen-containing
cryopaste:dissolution buffer=1 kg:4 L), and then stirred at a
controlled stirrer speed of 150 rpm at 37.degree. C. for 2
hours.
[0057] 3) The fibrinogen-containing cryopaste solution was
controlled to a temperature of 20 to 25.degree. C., and then 150 g
of aluminum hydroxide (Al(OH).sub.3) was added per kg of the
fibrinogen-containing cryopaste (preferably, 2% aluminum hydroxide
was added at a proportion of 0.5% by weight), followed by stirring
at a controlled stirrer speed of 150 rpm at 20 to 25.degree. C. for
1 hour.
[0058] 4) After completion of the reaction, centrifugation was
performed at 4000 rpm at 15.degree. C. for 30 minutes.
[0059] 5) After completion of the centrifugation, the supernatant
was filtered through the Opticap XL2 Polysep II filter (Merck
Millipore).
[0060] Virus Inactivation Process: Solvent/Detergent (S/D)
Treatment
[0061] 55.7 mL of virus inactivation solution (1% Tween 80, 0.3%
TNBP (tri-N-butyl phosphate)) was added per 1 L of the filtered
fibrinogen-containing cryopaste solution, followed by stirring at
150 rpm at 25.degree. C. for 1 hour.
[0062] As a result, a solution in which putative virus components
were inactivated was obtained by treating the filtered
fibrinogen-containing cryopaste solution with solvent/detergent
(S/D).
Example 2: 1.sup.st Glycine Precipitation Process
[0063] 1) To the putative virus component-inactivated, filtered
fibrinogen-containing cryopaste solution of Example 1, glycine was
added until a final concentration of the glycine concentration
becomes 1.9 to 2 M. At this time, the glycine was added so that the
protein content of the fibrinogen-containing cryopaste solution
would be 20 to 30 mg/mL.
[0064] 2) Stirring was performed at a controlled stirrer speed of
300 rpm at 4 to 25.degree. C. for 90 minutes.
[0065] 3) The stirred, glycine-added, fibrinogen-containing
cryopaste solution of 2) was centrifuged at 4000 rpm at 15.degree.
C. for 30 minutes.
[0066] 4) The supernatant was removed, and the precipitate
(hereinafter referred to as 1.sup.st glycine precipitation
precipitate) was weighed, and then freeze-stored at -70.degree. C.
or below.
Example 3: 2.sup.nd Glycine Precipitation Process
[0067] 3-1: 2.sup.nd Glycine Precipitation Process (Conditions
Under 20.degree. C., 0.75M and 15 mg/mL)
[0068] 1) To dissolve the freeze-stored 1.sup.st glycine
precipitation precipitate of Example 2, dissolution buffer (50 mM
sodium citrate, pH 7.5) was placed in a jacketed beaker, and then
stirred at a controlled circulator temperature of 37.degree. C. for
30 minutes or more. At this time, the temperature of the buffer was
maintained at 37.degree. C.
[0069] 2) The freeze-stored 1.sup.st glycine precipitation
precipitate of Example 2 was added to the dissolution buffer of 1),
and then stirred at a controlled stirrer speed of 150 rpm at
37.degree. C. for 90 minutes.
[0070] 3) The stirred 1.sup.st glycine precipitation precipitate
solution of 2) was filtered through the Opticap XL2 Polysep II
filter (Merck Millipore).
[0071] 4) The filtered 1.sup.st glycine precipitation precipitate
solution of 3) was placed in a jacketed beaker, and then the
temperature of the circulator was controlled to 20.degree. C.
[0072] 5) When the 1.sup.st glycine precipitation precipitate
solution reached a predetermined temperature, glycine was added
until a final concentration of the glycine concentration becomes
0.5 to 0.9M. At this time, the glycine as added so that the protein
content of the 1.sup.st glycine-added precipitation precipitate
solution would be 14 to 15 mg/mL.
[0073] 6) Stirring was performed at 300 rpm for 90 minutes while
the temperature of the circulator was maintained at 20.degree.
C.
[0074] 7) The stirred, glycine-added 1.sup.st glycine precipitation
precipitate solution of 6) was centrifuged at 4000 rpm at
15.degree. C. for 30 minutes.
[0075] 8) The precipitate of 7) was removed, and the amount of the
supernatant (hereinafter referred to as 2.sup.nd glycine
precipitation supernatant) was measured.
[0076] 3-2: 2.sup.nd Glycine Precipitation Process (Conditions
Under 25.degree. C., 0.8 M and 20 mg/mL)
[0077] An experiment was performed in the same manner as described
in Example 3-1, except that the following conditions were used:
glycine concentration: 0.8M; temperature: 25.degree. C.; and
protein concentration: 19 to 20 mg/mL.
[0078] 3-3: 2.sup.nd Glycine Precipitation Process (Conditions
Under 15.degree. C., 0.8M and 10 mg/mL)
[0079] An experiment was performed in the same manner as described
in Example 3-1, except that the following conditions were used:
glycine concentration: 0.8M; temperature: 15.degree. C.; and
protein concentration: 9 to 10 mg/mL.
[0080] 3-4: 2.sup.nd Glycine Precipitation Process (Conditions
Under 15.degree. C., 0.5M and 20 mg/mL)
[0081] An experiment was performed in the same manner as described
in Example 3-1, except that the following conditions were used:
glycine concentration: 0.5M; temperature: 15.degree. C.; and
protein concentration: 19 to 20 mg/mL.
Example 4: 3.sup.rd Glycine Precipitation Process
[0082] 1) The temperature of the circulator was controlled to
20.degree. C. When the temperature of the 2.sup.nd glycine
precipitation supernatant obtained in Example 3 reached 20.degree.
C., glycine was added to the supernatant until a final
concentration of the glycine concentration becomes 2.1M.
[0083] In a comparative example, the 1.sup.st glycine precipitation
precipitate obtained in Example 2 was dissolved in 50 mM sodium
citrate (pH 7.5) buffer at 35.degree. C. for 90 minutes.
Thereafter, glycine was added to the solution until a final
concentration of the glycine concentration becomes 2.1M.
[0084] 2) Stirring was performed at 300 rpm for 90 minutes.
[0085] 3) The stirred, glycine-added, 2.sup.nd glycine
precipitation supernatant of 2) was centrifuged at 4000 rpm at
15.degree. C. for 30 minutes.
[0086] 4) The supernatant was removed, and the precipitate
(hereinafter referred to as 3.sup.rd glycine precipitation
precipitate) was weighed, and then freeze-stored at -30.degree. C.
or below.
[0087] Analysis of Sample Purity by SEC-LC
[0088] The purity of fibrinogen in the 3.sup.rd glycine
precipitation precipitate, subjected to the 2.sup.nd glycine
precipitation process, and the purity of fibrinogen in the 3.sup.rd
glycine precipitation precipitate not subjected to the 2.sup.nd
glycine precipitation process were measured by the following test
method and compared with each other.
[0089] a. Sample solution preparation: the sample concentration was
corrected to 2 mg/mL, and 200 .mu.L of the sample was added to each
LC vial.
[0090] b. Test conditions: a test was performed under the following
conditions: flow rate: 0.5 mL/min; column temperature: room
temperature (RT); injection volume: 20 .mu.L; analysis time: 30
minutes; detection wavelength: UV 280 nm; and pump distribution:
100% isocratic.
[0091] c. Test Procedure:
[0092] 1) In accordance with Waters HPLC operating instructions, an
HPLC system was prepared.
[0093] 2) A TSK-gel G3000SW.sub.XL column and a TSKgel SWXL guard
column were disposed along the liquid flow direction.
[0094] 3) Equilibration of column: a mobile phase was allowed to
flow at a flow rate of 0.5 mL/min for at least 30 minutes, and when
the equilibration on the chromatogram was reached, the flow was
stopped.
[0095] 4) Analysis was performed under the same conditions as the
above-described test conditions.
[0096] Table 1 below shows the purity of fibrinogen obtained when
the 2.sup.nd glycine precipitation process was performed or not
performed.
[0097] In addition, FIG. 3 shows the results obtained when
performing only the 1.sup.st and 3.sup.rd glycine precipitation
processes without the 2.sup.nd glycine precipitation process, and
FIGS. 4 (conditions of Example 3-1), 5 (conditions of Example 3-2),
6 (conditions of Example 3-3) and 7 (conditions of Example 3-4)
shows the results obtained when performing all the 1.sup.st to
3.sup.rd glycine precipitation processes.
[0098] In summary, as shown in Table 1 below and FIGS. 3 to 7, when
all the 1.sup.st to 3.sup.rd glycine precipitation processes were
performed, the purity of the finally obtained fibrinogen was
significantly high regardless of the conditions of the
precipitation processes, compared to when only the 1.sup.st and
3.sup.rd glycine precipitation processes without the 2.sup.nd
glycine precipitation process were performed. Thus, it could be
seen that when the precipitation processes are performed using
glycine at lowered concentrations, the effect of removing
impurities produced from the virus inactivation process is
obtained.
TABLE-US-00001 TABLE 1 polymer or Purification impurity Fibrinogen
No. method (%) (%) Remarks 1 Examples 2 & 4 5.7 94.21 Example 3
not performed performed 2 Examples 2, 3 1.96 98.04 Example 3-1
& 4 performed performed 3 Examples 2, 3 1.64 98.25 Example 3-2
& 4 performed performed 4 Examples 2, 3 1.33 98.67 Example 3-3
& 4 performed performed 5 Examples 2, 3 1.77 98.23 Example 3-4
& 4 performed performed
Example 5: Optional 4.sup.th Glycine Precipitation Process
[0099] It was shown that when the 1.sup.st to 3.sup.rd glycine
precipitation processes as described in Examples 1 to 4 above were
all performed, fibrinogen could be obtained with very high purity.
In this Example, investigation was performed to determine whether,
when an additional glycine precipitation process which is not an
essential process was optionally performed, the purity of
fibrinogen could be further increased.
[0100] 1) To dissolve the freeze-stored 3.sup.rd glycine
precipitation precipitate of Example 4, dissolution buffer (50 mM
sodium citrate (pH 7.5)) was placed in a jacketed beaker, and then
stirred at a controlled circulator temperature of 37.degree. C. for
30 minutes.
[0101] 2) The freeze-stored 3.sup.rd glycine precipitation
precipitate of Example 4 was added to the buffer of 1), and then
stirred at a controlled stirrer speed of 290 to 310 rpm at 30 to
35.degree. C. for 90 minutes.
[0102] 3) The temperature of the 3.sup.rd glycine precipitation
precipitate solution was lowered to 25.degree. C., and glycine was
added to the solution to a final concentration of 2.1M.
[0103] 4) Stirring was performed at 300 rpm for 90 minutes while
the temperature of the circulator was maintained at 25.degree.
C.
[0104] 5) The stirred, glycine-added 3.sup.rd glycine precipitation
precipitate solution was centrifuged at 4000 rpm at 15.degree. C.
for 30 minutes.
[0105] 6) The 4.sup.th glycine precipitation precipitate of 5) was
recovered, weighed, and then freeze-stored at -30.degree. C. or
below.
Example 6: DoE Experiment on Nano-Filtration Process
[0106] 1) 250 mg or more of the 3.sup.rd or 4.sup.th glycine
precipitation precipitate obtained in Example 4 or 5 was
prepared.
[0107] 2) Dissolution buffer (containing 20 mM sodium citrate at pH
7.5, 100 mM sodium chloride (NaCl), and 1 to 6% arginine) was added
to the 3.sup.rd or 4.sup.th glycine precipitation precipitate, and
the precipitate was dissolved by stirring at room temperature (20
to 25.degree. C.) for 2 hours so that the final protein
concentration would be about 3 mg/mL.
[0108] 3) The concentration of the dissolved 4.sup.th glycine
precipitation precipitate of 2) was measured at an UV wavelength of
280 nm, and then the precipitate was diluted to a concentration of
0.7-3 mg/mL by use of dissolution buffer (containing 20 mM sodium
citrate at pH 7.5, 100 mM sodium chloride (NaCl), and 1 to 6%
arginine), and the final concentration was measured at a UV
wavelength of 280 nm.
[0109] 4) After correction of concentration, the sample was
filtered through a bottle top filter (0.2 .mu.m).
[0110] 5) The filtered sample of 4) was filtered through a Pall
100N filter (10 cm.sup.2 area) at room temperature (20 to
25.degree. C.) and a pressure of 2 bar.
[0111] 6) The filtered sample of 5) was filtered again through a
Pall 50N (or Planova 35N) filter (10 cm.sup.2 area). At this time,
the filtration was performed under the conditions of room
temperature (20 to 25.degree. C.) and 2 bar (1 bar for Planova
35N).
[0112] 7) The filtered sample of 6) was filtered through a Planova
20N (or SV4 20N) (10 cm.sup.2 area). At this time, the filtration
was performed under the conditions of 25 to 35.degree. C. and 0.5
to 2 bar (Planova 20N: 0.5 to 1 bar; SV4 20N: 1 to 2 bar)
(nano-filtration process time: 15 to 20 hours) (after Example 6 is
performed, UF/DF as an additional process may be performed).
[0113] Table 2 below shows the efficiency and recovery rate of the
Planova 20N and SV4 20N filter, obtained when the 2.sup.nd glycine
precipitation process was performed or not performed.
TABLE-US-00002 TABLE 2 Planova 20N (Asahi) Pall SV4 20N (Pall) 94%
Purity 98% Purity 94% Purity 98% Purity Efficiency 105.4 203.8
145.9 451.2 of filter (g/m.sup.2) Recovery of 76.1 92.3 90.5 92.1
filter (%)
[0114] As a result, as shown in Table 2 above and FIG. 8, the
purity of fibrinogen filtered through the nano-filter was compared
between cases that the 2.sup.nd glycine precipitation process was
performed and not performed as described in Example 3. As a result
of the comparison, it was shown that when the 2.sup.nd glycine
precipitation process was performed, the recovery of fibrinogen
with a purity of 98% was high. Therefore, when the precipitation
processes are performed using glycine at lowered concentrations,
the effects of removing impurities produced in the virus
inactivation process and increasing the filtration efficiency of
the nano-filter at least 2-fold were obtained.
INDUSTRIAL APPLICABILITY
[0115] The novel method for purifying fibrinogen protein according
to the present disclosure is a highly safe method that
fundamentally blocks the entry of pathogenic components derived
from animals (viruses). Furthermore, it can purify fibrinogen
protein with high purity and high recovery rate by a simple
process, and thus is very economical and efficient. In addition,
fibrinogen purified by the method of the present disclosure has an
advantage over fibrinogen purified by a conventional method in that
it has higher purity, and thus has increased local activity,
indicating that it can be effectively used for the prevention and
treatment of blood-related diseases, particularly blood clotting
diseases.
[0116] Although the present disclosure has been described in detail
with reference to the specific features, it will be apparent to
those skilled in the art that this description is only for a
preferred embodiment and does not limit the scope of the present
disclosure. Thus, the substantial scope of the present disclosure
will be defined by the appended claims and equivalents thereof.
* * * * *